This invention relates to detecting a pathogenic fungus, Histoplasma capsulatum, using oligonucleotide probes specific for H. capsulatum to amplify H. capsulatum DNA by means of the polymerase chain reaction. Test samples may originate from the environment, where H. capsulatum spores are found, or from clinical samples obtained from patients.
Histoplasma capsulatum is a dimorphic fungus that is pathogenic in humans. In one form, the fungus has the mycelia characteristic of molds, and produces spores as an aspect of its reproduction. This form is commonly found in the ambient environment, and is highly prevalent in soils containing guano from bats or birds. In general it proliferates in soils rich in nutrients such as those containing rotting organic residues. Its morphology includes multicellular filaments containing cylindrical structures (i.e., the hyphae or conidia). This form may further contain macroconidia and microconidia which may grow in soil or on media designed for fungal growth at 25xc2x0 C.
In its second form, H. capsulatum adopts a single celled state characteristic of yeast, and reproduces by budding. This form is prevalent on suitable nutrient media in the laboratory, and is the form generally found after it infects a human. Infection may occur upon breathing dust containing fragments of H. capsulatum hyphae or conidia; as noted above, such fragments may be prevalent if dust is stirred up from soil containing the droppings from birds or bats or soil that is otherwise rich in organic nutrients, especially nitrogenous nutrients.
The infection produces a systemic fungal disease, histoplasmosis, which can initially result in a variety of symptoms often dependent on the type of infection, the general health of the host, and the exposure level. In acute self-limiting infections, especially those occurring in healthy individuals, the symptoms may be very mild, such as those of a flu-like respiratory illness, with symptoms such as general malaise, fever, chest pain, a dry cough, headache, loss of appetite, shortness of breath, joint and muscle pains and chills. The severity of the symptoms may depend on how many spores have been inhaled, and the general health of the subject.
The symptoms of respiratory infection are often suppressed by the immune response. This response includes endocytosis by pulmonary macrophages. H. capsulatum may then proliferate within macrophages and be transported systemically as a result. As a result, such suppression appears to result in long term latency rather than in elimination of the organism. H. capsulatum infection can also result in calcified deposits within the lungs, produced when growth of the yeast is inhibited by host immune responses. Both the infected macrophages and the calcified deposits serve as a reservoir of latent infection. As a consequence, if the individual subsequently becomes immune compromised or immune deficient, histoplasmosis can reappear. Thus individuals such as those receiving immune-suppressing therapy, the very young or the elderly, those suffering from cancer, or expressing the symptoms of acquired immune deficiency syndrome are susceptible to recrudescence of histoplasmosis as a chronic and/or a disseminated infection, and may include ocular infection. Resurgence of such latent histoplasmosis produces potentially lethal symptoms.
Assay for H. capsulatum infection or its presence in a sample is carried out by methods such as culturing, immunoassay for surface antigens, and a bioassay. Currently, histoplasmosis may be diagnosed by culture, or by the demonstration of a rise in complement-fixing antibody titers in serum. A definitive diagnosis of an H. capsulatum infection requires the isolation and propagation of the fungus, which is time-consuming and lacks sensitivity. It is also dangerous for laboratory personnel, who must take extreme caution to prevent inhalation of the pathogenic fungus, so as not to become ill with a pulmonary infection. Further, only small quantities of antigens of H. capsulatum for use as biological reagents may be prepared in this manner.
Conventional laboratory identification methods used to isolate and identify H. capsulatum include the culture of a clinical specimen at room temperature on specialized fungal media. This procedure isolates the slower growing H. capsulatum colonies from possible contaminants, such as bacteria, and from faster growing saprobic fungi. This method, however, has several disadvantages. Because the growth of H. capsulatum to a visible colony normally takes from about two to four weeks, and sometimes as long as 12 weeks, this identification procedure is very slow. (See, for example, Rippon, Histoplasmosis. In Medical Mycology, The Pathogenic Fungi and the Pathogenic Actinomyestes, 3rd Ed., Saunders, Ch. 15 (1988); Koneman at al., Laboratory Identification of Molds, in Practical Laboratory Mycology, (3rd ed. Williams and Wilkins (1985)); and McGinnis, Histoplasma capsulatum, in Laboratory Handbook of Medical Mycology, Academic Press (1986).) Further, additional growth is required before the characteristic colony morphology and microscopic sporulation pattern with tuberculate macroconidia may be observed. In addition, approximately 10% of cultures produce only smooth-walled macroconidia, and some cultures fail to sporulate. Moreover, many species of fungi other than H. capsulatum, such as Blastomyces dermatitides, Chryacsporium sp., and Sepedonium sp., produce similar colony and sporulation characteristics. Thus, additional testing is usually necessary to definitively identify the organism.
One method of converting the mycelial colony of H. capsulatum to the yeast phase is performed by subculturing the organism onto highly enriched cysteine-containing media, and incubating it at 35-37xc2x0 C. However, conversion to the yeast phase is often difficult, and may require several additional subcultures at three-day intervals.
Histoplasmin, an unpurified culture supernatant obtained from the mycelial phase of H. capsulatum contains H. capsulatum M antigens. Histoplasmin is currently used to probe both humoral and cell-mediated responses in patients with histoplasmosis. It is used for the serologic diagnosis of histoplasmosis, and as a skin test antigen to demonstrate delayed hypersensitivity to infection in skin tests for histoplasmosis. The purification of histoplasmin is described by Bradley et al., Infect. Immun. 9:870-880 (1974). The preparation of H and M antigens of H. capsulatum free of heterologous antigens is described by Green et al., Curr. Microbiol. 12:209-216 (1985). (See also, Pine, Contrib. Microbiol. Immunol. 3:138-168 (1977).) The preparation of antisera to the M antigen is described by Green et al., Infect. Immun. 14:826-831 (1976). General information concerning the serodiagnosis of fungal diseases is present in Kaufman et al., Serodiagnosis of Fungal Diseases, in Manual of Clinical Laboratory Immunology (3rd ed., American Society for Microbiology, Washington, D.C.(1988)).
Serologic evidence is a principal diagnostic indicator of histoplasmosis. Several serologic tests, such as the immunodiffusion test, detect precipitants against the species-specific H and M antigens found in histoplasmin. (See, for example, Kaufman, Clin. Infect. Dis. 14:23-29 (1992), and Wheat, Eur. J. Clin. Microbiol. Infect. Dis. 8:480 (1989).)
Although the M antigen of H. capsulatum is useful in immunoassays for the diagnosis of histoplasmosis, purification of the M antigen from a batch culture is a laborious and low-yield process. The use of a recombinantly-produced M antigen of H. capsulatum in such immunoassays would significantly diminish the labor necessary to obtain M antigens which are pure enough to be useful in the immunoassays, and would result in high yields of the M antigen.
Assay for H. capsulatum infection historically has been via immunoassay for surface antigens. Additionally, a widely practiced assay is the mouse inoculum/agar plate culture method. This method, which involves indirect detection, takes as long as 6-8 weeks and is expensive. The method entails extracting a soil sample suspected of harboring H. capsulatum and injecting the extract intraperitoneally into mice. After six weeks the mice are sacrificed, and extracts of the livers and spleens are inoculated onto Sabouraud""s dextrose agar. These are incubated and observed weekly for fungal growth. Any fungi resulting are then subcultured for a more definitive identification. (Ajello, L. and Weeks, R. J., in Occupational Mycoses, A Text, edited by A. F. DiSalvo, Leas and Febiger, Philadelphia, pp.229-238, 1983; Ajello, L. and Runyon, L. C., J. Bacteriol. 66:34-40, 1953). More recently molecular biological assay techniques have been developed, such as restriction fragment length polymorphism (RFLP).
U.S. Pat. No. 5,580,971 to Mitsuhashi discloses methods for detecting a particular fungus in a biological sample based on polynucleotide capture probes. In one method, the probe is complementary to a sequence in the ribosomal RNA of a particular fungus, and binding of the analyte nucleic acid is detected with a second probe whose sequence is common to a wide range of fungi. In another method, two probes are used and nucleic acid is amplified using, preferably, a polymerase whose activity is not hindered at high temperature. Many species and genera of pathogenic fungi are detectable, but H. capsulatum is not included in the disclosed group of fungi.
WO 96 19588 discloses a nucleic acid hybridization probe derived from the saccharopine dehydrogenase of Candida albicans that is conserved in fungi. The probe, and an assay method employing the probe, may be used for detection of a variety of fungi from different genera. WO 96 21741 discloses oligonucleotide probes considered to be universal primers for 28S rRNA of fungi. An extensive range of species and genera of fungi is disclosed as being detectable by amplification methods using the probes, including H. capsulatum. 
Padhye et al. (J. Clin. Microbiol. 30, 3108-3111 (1992)) describe application of the Accuprobe(trademark) (Gen Probe, Inc., San Diego, Calif.) assay for H. capsulatum. Accuprobe is a DNA-based probe in which hybridization between the probe and RNA from the organism is detected by a chemiluminescent assay. 103 of 105 H. capsulatum samples were identified by the assay, with assay times of about 2 hrs. Hall (Arch. Pathol. Lab. Med. 117, 578-583 (1993)) reviews applications of the Accuprobe(trademark) assay in general, and summarizes the organisms for which Accuprobes(trademark) were available. H. capsulatum is included among these. Hall does not provide the structure of the H. capsulatum probe.
Spitzer et al. (Infect. Immun. 57, 1409-1412 (1989)) disclose classification of ambient and clinical isolates of H. capsulatum based on analysis of mitochondrial DNA and ribosomal DNA. Keath et al. (J. Clin. Microbiol. 30, 2104-2107 (1992)) present analyses of a large number of clinical and soil samples of H. capsulatum using restriction fragment length polymorphisms (RFLP). The nuclear gene probe yps-3 and a mitochondrial DNA probe were employed, and permitted classification into the known, as well as the identification of new, classes and subclasses of the fungus.
Kersulyte et al. (J. Bact. 174, 7075-7079 (1992)) show that a polymerase chain reaction-based amplification based on random primers drawn from the fungal genome, and RFLP analysis resolves various isolates of H. capsulatum into a range of strains. They find that H. capsulatum is an extremely diverse species. Woods et al. (J. Clin. Microbiol. 31, 463-464 (1993)) teach that use of randomly chosen primers for use in the polymerase chain reaction allows simple and rapid detection of variant isolates of H. capsulatum. They describe a rapid isolation method to obtain DNA from H. capsulatum in order to permit sensitive identification of H. capsulatum strains.
U.S. Pat. No. 5,352,579 to Milliman discloses probes for the detection of Histoplasma capsulatum which distinguishes between H. capsulatum and its known closest phylogenetically related organisms. The invention is based on a hybridization probe capable of effecting this distinction. The probe is complementary to a variable region of rRNA or rDNA. A method of assaying for H. capsulatum using this probe is also provided. Mayer et al. (96th General Meeting, Amer. Soc. for Microbiol., New Orleans, La., May 19-23, 1996) have devised a set of PCR probes (whose sequences were not disclosed) that are complementary to the gene for the H antigen of H. capsulatum var. capsulatum and var. duboisii that amplifies H. capsulatum DNA with specificity and sensitivity. They also disclose the use of a nucleotide probe in a TaqMan assay, wherein the probe hybridizes to a region of H. capsulatum between the binding sites of two PCR probes. In the assay Taq polymerase liberates a luminescent probe from a quenching environment if the probe binds target DNA.
Presently, the method used to isolate and identify H. capsulatum is expensive and requires several weeks to complete. The expense is increased by the number of samples required. If not enough samples are collected, small but highly contaminated areas can be overlooked. See, e.g., xe2x80x9cHistoplasmosis: Protecting Workers at Riskxe2x80x9d, Dept. of Health and Human Services Publication No. 97-146, September 1997. Therefore, in the current status of the field, there remains a need for a specific, sensitive, and rapid assay for H. capsulatum, both in environmental samples and in clinical samples from patients suspected of harboring H. capsulatum infection. The present invention provides such methods and assays.
The present invention provides oligonucleotides which are useful as primers to initiate the amplification of a segment of H. capsulatum DNA that is specific to H. capsulatum using the polymerase chain reaction. The present invention also provides methods of detecting the presence of H. capsulatum in a sample using a nested, or two-stage, PCR assay.
The invention discloses an oligonucleotide primer pair including sequences specific for the 5.8S rRNA gene of Histoplasma capsulatum, to be used in the second stage of the PCR assay, or an oligonucleotide primer pair, each of which includes a sequence complementary to one or the other of the primer pair specific for the H. capsulatum 5.8S rRNA gene. In a significant embodiment, the oligonucleotide primer pair is disclosed as an oligonucleotide containing the sequence given by SEQ ID NO:3, and an oligonucleotide containing the sequence given by SEQ ID NO:4. The invention additionally discloses an oligonucleotide containing a sequence complementary to that given in SEQ ID NO:3, and an oligonucleotide containing a sequence complementary to that given in SEQ ID NO: 4. These complementary oligonucleotides are useful to prepare the actual PCR primers of the invention by the action of a DNA polymerase, and in probing test samples for the presence of H. capsulatum DNA.
The methods of the invention detect the presence of Histoplasma capsulatum in a sample. One of these methods includes the steps of:
(a) providing a sample suspected of harboring H. capsulatum; 
(b) preparing a first amplified nucleic acid by contacting the sample with a first solution comprising an oligonucleotide primer pair specific for a fungal nucleic acid sequence that comprises the coding sequence for the 5.8S rRNA gene, a first DNA polymerase, and a first mixture of deoxynucleotide triphosphates, in a first buffer, and carrying out a polymerase chain reaction on the first solution resulting therefrom;
(c) preparing a second amplified nucleic acid by contacting all or a portion of the first amplified nucleic acid from step (b) with a second solution comprising an oligonucleotide primer pair specific for the H. capsulatum 5.8S rRNA gene, a second DNA polymerase, and a second mixture of deoxynucleotide triphosphates, in a second buffer, and carrying out a polymerase chain reaction on the second solution resulting therefrom; and
(d) detecting the presence of DNA derived from H. capsulatum in the second amplified nucleic acid,
whereby the presence of DNA derived from H. capsulatum in the second amplified nucleic acid indicates that H. capsulatum occurs in the sample. In a preferred embodiment of the above method, the oligonucleotide primer pair employed in step (b) is specific for the fungal internal transcribed spacer (ITS) regions flanking the coding sequence for the 5.8S rRNA gene (see FIG. 1).
The invention additionally provides a method of detecting the presence of Histoplasma capsulatum in a sample, including
(a) providing a sample suspected of harboring H. capsulatum; 
(b) preparing a first amplified nucleic acid by contacting the sample with a first solution comprising an oligonucleotide comprising the sequence given by SEQ ID NO:1, an oligonucleotide comprising the sequence given by SEQ ID NO:2, a first DNA polymerase, and a first mixture of deoxynucleoside triphosphates, in a first buffer, and carrying out a polymerase chain reaction on the first solution resulting therefrom;
(c) preparing a second amplified nucleic acid by contacting all or a portion of the first amplified nucleic acid from step (b) with a second solution comprising an oligonucleotide comprising the sequence given by SEQ ID NO:3, an oligonucleotide comprising the sequence given by SEQ ID NO:4, a second DNA polymerase, and a second mixture of deoxynucleoside triphosphates, in a second buffer, and carrying out a polymerase chain reaction on the second solution resulting therefrom; and
(d) detecting the presence of DNA derived from H. capsulatum in the second amplified nucleic acid,
whereby the presence of DNA derived from H. capsulatum in the second amplified nucleic acid indicates that H. capsulatum occurs in the sample.
In preferred embodiments of the methods of the invention the samples can be environmental samples, including soil samples, or clinical samples from human subjects. Furthermore, in a preferred embodiment of the methods of the invention, the partial purification step includes separating any soil contained in the sample from the H. capsulatum nucleic acid. In still another preferred embodiment of the invention, the first buffer contains bovine serum albumin. In additional preferred embodiments of the methods the detecting step includes gel electrophoresis of the second amplified nucleic acid and detecting any H. capsulatum-specific DNA in the resulting gel, or contacting the second amplified nucleic acid with a labeled probe specific to H. capsulatum-specific DNA and detecting the label.